The respiratory pathogens Mycobacterium tuberculosis, Chlamydia pneumoniae, and Legionella pneumophila all have an obligate intracellular lifestyle that requires establishment of a replicative vacuole that does not fuse with lysosomes. Despite the fact that creation of a non-degradative vacuole is an essential virulence trait, very little is known about the molecular mechanisms that govern biogenesis of the replicative organelles in which these bacteria reside. L. pneumophila has become a valuable model organism for identifying bacterial determinants that govern biogenesis of these specialized replicative organelles. The Dot/Icm (defective organelle trafficking, and intracellular multiplication) transporter is a protein secretion apparatus that is essential for L. pneumophila pathogenesis. This transporter has the ability to inject bacterial proteins into eukaryotic host cells. These proteins are predicted to regulate the formation of a vacuole that supports intracellular growth of L. pneumophila. The goal of this grant is to understand how this transporter functions. Towards this end we will identify bacterial and host factors that play important roles in biogenesis of a replicative vacuole. Specifically, we will: 1) identify and investigate functional domains in cytoplasmic Icm proteins that regulate distinct activities necessary for biogenesis of a replicative organelle, 2) investigate the role of DotA protein secreted by the Dot/Icm transporter, 3) characterize RalF protein, which is an ARF-specific guanine nucleotide exchange factor injected into host cells by the Dot/Icm transporter, 4) identify other substrates of the Dot/Icm transporter that are injected into host cells, 5) define cellular processes that are required for the establishment of a replicative organelle by L. pneumophila. Determining the molecular function of L. pneumophila gene products required for phagosome trafficking will provide us with a foundation for how bacterial pathogens are able to alter their intracellular fate and profoundly affect the development of new strategies for fighting diseases caused by bacteria that modify phagosome maturation.